The molecular interactions that mediate signaling of T and B lymphocytes, both during development and in activation of mature T and B cells, are not fully understood. Genetic and biochemical approaches have therefore been taken to address these questions. Costimulatory B7 molecules (B7-1 or CD80;and B7-2 or CD86) are known to bind to T cell costimulatory receptors CD28 and CTLA4. Engagement of CD28 is know to transduce signals in T cells that play a critical role in T cell activation. TCR mediated activation of T cells to proliferation and IL2 secretion was enhanced in mice deficient in the adapter molecule cbl-b, suggesting a negative regulatory role for cbl-b under these conditions. Cbl-b deletion in fact reversed the inability of CD28-deficient mice to generate T cell-dependent Ig class switched primary and secondary antibody responses. The observation that cbl-b inactivation can enhance T cell responses to TCR signaling, and can bypass requirements for CD28 costimulation has been translated to studies of tumor rejection. Inactivation of cbl-b enhances the ability of mice to reject tumors that do not express costimulatory ligands (CD80 or CD86), and that grow progressively in wild type mice. Cbl-b inactivation also dramatically inhibits the incidence of thymic lymphomas in ATM-deficient mice. These findings identify a novel strategy for enhancing T cell-mediated tumor immunotherapy by modulating regulatory influences on T cell activation. Translational efforts are in progress using high throughput screening to identfy molecules that are capable of reproducing the effects of cbl-b inactivation, allowing human T lymphocytes to respond to T cell receptor (TCR) stimuli in the absence of otherwise required CD28-mediate cosimulation. Mutants of B7-1 have been generated and studied that differ in their capacity to dimerize, potentially affecting their interaction with receptors such as CD28 and CTLA4. B7-1 mutants that are obligate covalent dimers are more efficient in supporting conjugates of T cells and antigen-presenting cells (APC) than is the wild-type B7-1 which exists in dynamic equilibrium between monomer and dimer. Obligate dimers are also more efficient in inducing early signaling events in T cells. Paradoxically, however, B7-1 dimers are less potent costimulators of T cell proliferative responses. These findings suggest that reversible transition between monomer and dimer may be critical in the optimal kinetics of interaction involved in costimulation of T cell functional responses necessary for functional responses to antigen, with potential relevance to responses to tumors and infectious agents. BAC transgenes encoding these altered forms of B7-1 have been constructed and will be used to generate transgenic mice, allowing in vivo analysis of structure-function relationships. The relationship between members of the cbl family and other critical adapter molecules has been studied. Experiments have indicated that inactivation of c-cbl (but not cbl-b) completely reverses the lethal phenotype caused by SLP-76 deficiency. In addition, c-cbl inactivation partially reverses the defect in T cell development caused by deficiency in SLP-76, LAT,or Vav1. These findings indicate an unanticipated SLP-76 (and LAT) independent signaling pathway that is facilitated by c-cbl inactivation. The biochemical basis for these effects has been studied using SLP-76 mutants to dissect the structure-function relationships in this pathway. C-cbl specifically rescues the T cell developmental defect resulting from SLP-76 mutations that disrupt tyrosines involved in binding to Vav and Itk. Subsequent analysis has further indicated that c-cbl inactivation rescues the developmental defect in Vav1-deficient mice, further characterizing a SLP-76/LAT/Vav1 pathway for T cell development. In contrast, c-cbl inactivation fails to overcome the T cell developmental defects that occur un lck-deficient mice. Thus, the pathways unmasked by c-cbl deficiency appear to require lck. The molecular components of this pathway are currently under study.